This Small Business Innovation Research (SBIR) Phase I project aims to make next generation sequencing technology for small RNA more quantitative and less biased. High throughput sequencing has transformed the landscape of genomic research with its ability to produce gigabases of data in a single run. This has enabled researchers to perform genome wide and high depth sequencing studies that would normally not be possible. Despite this capacity, amplification artifacts introduced during PCR increase the chance of duplicate reads and uneven distribution of read coverage. Accurate profiling using deep sequencing also has been undermined by biases with over or under-represented miRNAs. The presence of these biases significantly limits the incredible sensitivity and accuracy made possible by next generation sequencing. The goal of this proposal is to develop novel, bias-reducing technology for making amplification-free small RNA libraries. The company's kits and protocols will ramp-up considerably the rate at which global microRNA profiles can be determined, and that between-sample and within-sample differences (as well as newly discovered small RNAs) can be subsequently validated. This product will result in a major shift in the way small RNA sequencing is performed and pave the way for unbiased measurements in the clinic. The broader impact of this project will be the accurate measure of small RNAs, and the clinical utility of such a profile. Products of the same microRNA gene that vary in length by one or two nucleotides are involved in a whole host of diseases, including cancer. The value for developing a method to measure the true profile of microRNAs in a sample would be immense for the research community studying transcriptional regulation, and would open the doors to clinicians interested in capitalizing on the diagnostic value of microRNA profiling. Companies whose sole model is to extract prognostic information from microRNA profiles would benefit from the wealth of date generated from accurate non-biased high throughout sequencing. The size of the next generation sequencing market is expected to pass $4 billion by 2014. Growth in the sequencing diagnostic market is just beginning. Unique diagnostic kits developed from this technology will fulfill an unmet market opportunity with the potential to exceed $15 million in the first 3 years